本文選題：大片形吸蟲 + ES��； 參考：《黑龍江八一農(nóng)墾大學(xué)》2017年碩士論文

【摘要】：片形吸蟲不僅感染牛羊等反芻動(dòng)物,而且也可感染人體,嚴(yán)重威脅著人和動(dòng)物的健康。因此,建立高效準(zhǔn)確的診斷方法是防控該病的研究重點(diǎn)。片形吸蟲包括肝片形吸蟲、大片形吸蟲及其中間型,目前對于大片形吸蟲及其中間型的相關(guān)研究相對較少。因此,本研究選擇大片形吸蟲為研究對象,通過對大片形吸蟲排泄分泌(ES)抗原與宿主的互作,旨在篩選、鑒定及分析出具有診斷潛力的蛋白,為建立大片形吸蟲的診斷方法奠定基礎(chǔ)。本研究分為三部分。第一部分,對大片形吸蟲的鑒定。首先,將采自廣西南寧的144條片形吸蟲蟲體進(jìn)行形態(tài)學(xué)鑒定和分子生物學(xué)鑒定。形態(tài)學(xué)鑒定這144條蟲體均為片形吸蟲。對這144條蟲體的核糖體第二內(nèi)轉(zhuǎn)錄間隔區(qū)(ITS-2)序列進(jìn)行了擴(kuò)增、測序,結(jié)果表明144個(gè)PCR樣品的測序結(jié)果區(qū)別于肝片形吸蟲及中間型,而均與大片形吸蟲ITS-2序列(GenBank accession AJ557569)匹配率達(dá)99.7%,從而判定分離得到的144條片形吸蟲均為大片形吸蟲,不含中間型。第二部分,大片形吸蟲ES抗原的制備及互作抗原的篩選。首先,利用體外培養(yǎng)方法收集大片形吸蟲蟲卵,對蟲卵進(jìn)行體外培養(yǎng)孵出毛蚴感染淡水螺,使其在淡水螺體內(nèi)生長發(fā)育,直至尾蚴逸出螺體脫尾形成囊蚴,收集形成的囊蚴(感染蟲卵后35 d),并進(jìn)一步用囊蚴經(jīng)口感染水牛(約500個(gè)囊蚴/頭),在水牛感染后的14 d、42 d、70 d、98 d,分別采血制備血清。其次,利用體外培養(yǎng)方法分別收集大片形吸蟲ES抗原,將收集的ES抗原免疫家兔制備陽性血清,用間接ELISA方法檢測其抗體效價(jià),結(jié)果表明ES抗原制備的陽性血清效價(jià)可達(dá)到1:1 000,說明大片形吸蟲ES抗原的免疫原性較好,可用于后續(xù)的試驗(yàn)。然后,將大片形吸蟲ES抗原分別與水牛各時(shí)間段的血清互作,通過免疫共沉淀和LC-MS/MS(質(zhì)譜)試驗(yàn),共獲得了465個(gè)非冗余蛋白。進(jìn)一步用BlastP同源性比對、UniProt鑒定,找到57個(gè)已知蛋白,分別在42 d、70 d和98 d的蛋白有13(22.8%)個(gè)、5(8.8%)個(gè)和7(12.3%)個(gè),同時(shí)存在于42 d和70 d、70 d和98 d、42 d和98 d的可被鑒定的蛋白分別有8(14%)個(gè)、5(8.8%)、1(1.8%)個(gè),同時(shí)存在于42 d、70 d和98 d可被鑒定的蛋白有18(31.6%)個(gè)。進(jìn)一步用生物信息學(xué)分析,預(yù)測α-微管蛋白和亮氨酸氨肽酶等蛋白具有診斷價(jià)值。第三部分,對篩選出的具有代表性的α-微管蛋白和亮氨酸氨肽酶進(jìn)行克隆、原核表達(dá),并探索其作為片形吸蟲病診斷抗原的可行性。結(jié)果表明,成功克隆了片段長為1 356 bp、能編碼452個(gè)氨基酸、理論分子量為48.99 kDa的α-微管蛋白基因和片段長為1 572 bp、能編碼523個(gè)氨基酸、理論分子量為56.38 kDa的亮氨酸氨肽酶基因。這兩個(gè)蛋白的二級結(jié)構(gòu)預(yù)測都以無規(guī)卷曲為主,抗原表位在整個(gè)序列均有分布。三級結(jié)構(gòu)的主要功能區(qū)域都在N端。將α-微管蛋白基因插入載體pGEX-6P-1(+),亮氨酸氨肽酶基因插入載體pET-30a分別進(jìn)行原核表達(dá)。發(fā)現(xiàn)經(jīng)IPTG誘導(dǎo)表達(dá)的重組蛋白pGEX-6P-1(+)-FgAT分子量約為76 kDa(含載體攜帶的26 kDa的GST標(biāo)簽),且以融合蛋白形式表達(dá),經(jīng)Glutathione-Sepharose beads純化后,獲得了較高純度的重組蛋白pGEX-6P-1(+)-FgAT;經(jīng)IPTG誘導(dǎo)表達(dá)的重組蛋白pET-30a-FgLAP分子量約為56 kDa,以包涵體形式表達(dá),經(jīng)Ni-NTA His bind Resin純化后獲得較高純度的重組蛋白pET-30a-FgLAP。這兩個(gè)重組蛋白分別與大片吸蟲ES抗原免疫家兔后制備的陽性血清、水牛感染大片吸蟲不同期的陽性血清反應(yīng),經(jīng)Western Blot分析證實(shí)均具有免疫反應(yīng)性。本研究通過以上三個(gè)部分的試驗(yàn),成功鑒定出大片形吸蟲ES抗原中與宿主互作的蛋白,包括465個(gè)非冗余蛋白。利用生物信息學(xué)分析,篩選出57個(gè)已知蛋白,并進(jìn)一步鑒定出具有代表性的α-微管蛋白基因和亮氨酸氨肽酶基因。通過對這兩個(gè)基因的克隆、表達(dá)及免疫反應(yīng)性分析,推斷出它們在大片形吸蟲病診斷中具有很大的潛力。
[Abstract]:Lamellar flukes not only infect ruminants such as cattle and sheep, but also can infect human beings, which seriously threaten the health of people and animals. Therefore, the establishment of an efficient and accurate diagnosis method is the key point of prevention and control of the disease. The study is relatively small. Therefore, in this study, large tracts are selected as the research object. Through the interaction between ES antigen and the host, this study aims at screening, identifying and analyzing the diagnostic potential proteins to establish a diagnostic method for large tracts. This study is divided into three parts. Identification of insects. First, 144 flake bugs collected from Nanning, Guangxi, were identified by morphological identification and molecular biological identification. Morphological identification of these 144 worms were lamellar bugs. The sequence of the ribosome second internal transcriptional interval (ITS-2) sequence of the 144 worm bodies was amplified and sequenced, and the results showed that the sequence of 144 PCR samples was sequenced. Different from the liver Fasciola and the intermediate type, and the matching rate of the ITS-2 sequence (GenBank accession AJ557569) with the large tracts of Fasciola (accession AJ557569) is up to 99.7%. Thus, it is found that all of the 144 flake like flukes are large tracts, without intermediate type. Second, the preparation of ES antigen of large Fasciola and the screening of mutual antigen. First, the culture method is used in vitro The eggs were collected and cultured in vitro, and the eggs were incubated in vitro to infect freshwater snails, to grow and develop in fresh water spire, until the cercariae escaped from the spire to form the cycariae, and the cycariae were collected (35 d after the infection of the eggs), and the buffalo (about 500 cysts / heads) infected with the cysts, 14 d, 42 d, 7 after buffalo infection. 0 d and 98 D were collected to prepare blood serum respectively. Secondly, using the in vitro culture method to collect the large tracer ES antigen, the collected ES antigen was immunized to prepare the positive serum, and the antibody titer was detected by the indirect ELISA method. The results showed that the positive serum titer of the ES antigen preparation could reach 1:1 000, indicating the immunogen of the large Fasciola ES antigen. 465 non redundant proteins were obtained by immunoprecipitation and LC-MS/MS (mass spectrometry) test, and 57 known proteins were found in 42 d, 70 D and 98 D proteins, respectively, with BlastP homology comparison and UniProt test. There are 13 (22.8%), 5 (8.8%) and 7 (12.3%), which exist at 42 d and 70 D, 70 D and 98 D, 42 d and 98 D, respectively. The protein has diagnostic value. In the third part, we clone the representative alpha microtubulin and leucine aminopeptidase, prokaryotic expression, and explore its feasibility as a diagnostic antigen for flake like worm disease. The results showed that the fragment was successfully cloned to 1356 BP and could encode 452 amino acids, and the theoretical molecular weight was 48.99 kDa. The alpha microtubulin gene and fragment length are 1572 BP, which can encode 523 amino acids and a leucine aminopeptidase gene with a theoretical molecular weight of 56.38 kDa. The two structure prediction of these two proteins is dominated by random curling, and the epitope of the antigen is distributed throughout the sequence. The main functional areas of the three stage structure are at the N end. PGEX-6P-1 (+) and leucine aminopeptidase gene inserted into the vector pET-30a to express the prokaryotic expression. The molecular weight of the recombinant protein pGEX-6P-1 (+) -FgAT induced by IPTG was about 76 kDa (including the GST tag carried by the carrier), and expressed in the form of the fusion protein. The purity of the recombinant protein was purified by Glutathione-Sepharose beads, and the high purity was obtained. The recombinant protein pGEX-6P-1 (+) -FgAT, the molecular weight of recombinant protein pET-30a-FgLAP induced by IPTG was about 56 kDa, expressed in the form of inclusion body, and purified by Ni-NTA His bind to obtain a high purity recombinant protein pET-30a-FgLAP., the two recombinant proteins, which were immunized with the positive sera of the large Fasciola ES antigen to immunize rabbits respectively. Cattle infected with positive sera from different stages of Fasciola were immunoreactive by Western Blot analysis. This study successfully identified the proteins that interacted with the host, including 465 non redundant proteins, in the three parts of the test, and 57 known proteins were screened by bioinformatics analysis. Further identification of the representative alpha microtubulin gene and leucine aminopeptidase gene is further identified. By cloning, expressing and immunoreactive analysis of these two genes, it is concluded that they have great potential in the diagnosis of large tracepimiasis.

【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.7

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